Sialylation of cell surface glycoconjugates is essential for osteoclastogenesis

Sialic acid, which is located at the end of the carbohydrate moiety of cell surface glycoconjugates, is involved in many biologic responses, such as intercellular reactions and virus-cell fusion, especially in hematopoietic cells. Here we provide experimental evidence that the sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation. Lectin histochemical study demonstrated the existence of both alpha (2,3)-linked-sialic acid and alpha (2,6)-linked-sialic acid in mouse bone marrow-derived macrophages and in the RAW264.7 macrophage cell line, which are osteoclast precursors. Flow cytometric analysis of surface lectin staining revealed the kinetics of these sialic acids during osteoclastogenesis: alpha (2,3)-linked-sialic acid was abundantly expressed throughout osteoclastogenesis, whereas alpha (2,6)-linked-sialic acid levels declined at the terminal stage of osteoclast differentiation. To investigate the role of sialic acid in osteoclast differentiation, we performed an osteoclastogenesis assay with or without exogenous sialidase treatment. Desialylated cells formed TRAP-positive mononuclear cells, but did not become multinuclear cells despite the normal expression of osteoclast markers such as cathepsin K, integrin beta3, and nuclear factor-ATc1. Flow cytometric analysis also demonstrated that exogenous sialidase effectively removed alpha (2,6)-linked-sialic acid, but only slightly changed the alpha (2,3)-linked-sialic acid content, suggesting that alpha (2,6)-linked-sialic acid might be involved in osteoclast differentiation. Findings from knockdown analysis using small interfering RNA oligonucleotides against alpha 2,6-sialyltransferase support this idea: alpha (2,6)-linked-sialic acid-deficient cells markedly inhibit the formation of multinuclear osteoclasts. Our findings suggest that alpha (2,6)-linked-sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation, possibly via its role in the cell-cell fusion process.     

TRAF2 is essential for TNF-alpha-induced osteoclastogenesis.

TRAF2-deficient mice show embryonic lethality, and we developed a new in vitro differentiation system to show the function of TRAF2 in osteoclastogenesis, in which osteoclast progenitors are derived from the fetal liver of TRAF2-deficient mice. Using this system, we showed that TRAF2 is required for TNF-alpha-induced osteoclastogenesis. INTRODUCTION: TNF receptor-associated factor 2 (TRAF2) is a signal transducer for RANK and for two TNF receptor isotypes, TNFR1 and TNFR2. Because TRAF2-deficient mice show embryonic lethality, it has remained unclear whether TRAF2 is crucial in RANKL- or TNF-alpha-induced osteoclastogenesis. MATERIALS AND METHODS: Osteoclast progenitors derived from fetal liver were cultured in the presence of monocyte macrophage colony-stimulating factor (M-CSF), and flow cytometry for characterization of surface markers on these cells was performed. To examine the involvement of TRAF2 in osteoclast differentiation, we cultured osteoclast progenitors from TRAF2-deficient and wildtype mice with soluble RANKL or TNF-alpha in the presence of M-CSF, and counted the number of TRACP(+) multinucleate cells formed. c-jun N-terminal kinase (JNK) and NF-kappaB activation in osteoclast progenitors was examined by Western blot analysis and electrophoretic mobility shift assay, respectively. Nuclear factor of activated T cells (NFATc1) expression and activation were analyzed by RT-PCR and immunofluorescence staining, respectively. To examine whether TRAF2 overexpression induced osteoclastogenesis, TRAF2 was overexpressed in osteoclast progenitors form wildtype bone marrow by retrovirus infection. RESULTS AND CONCLUSIONS: Osteoclast progenitors from normal fetal liver, which were cultured with M-CSF, expressed surface molecules c-fms, Mac-1, and RANK, and could differentiate into TRACP(+) multinucleate cells in the presence of soluble RANKL or TNF-alpha. RANKL-induced osteoclastogenesis gave a reduction of 20% in the progenitors from TRAF2-deficient mice compared with that of the cells from littermate wildtype mice, whereas TNF-alpha-induced osteoclastogenesis was severely impaired in the cells from the TRAF2-deficient mice. Only a few TRACP(+) multinucleate cells were formed, and TNF-alpha-mediated activation of JNK, NF-kappaB, and NFATc1 was defective. TRAF2 overexpression induced differentiation of osteoclast progenitors from wildtype mice into TRACP(+) multinucleate cells. These results suggest that TRAF2 plays an important role in TNF-alpha-induced osteoclastogenesis.     

E-cadherin is important for cell differentiation during osteoclastogenesis

E-cadherin, a protein responsible for intercellular adhesion between epithelial cells, is also expressed in the monocyte/macrophage lineage. In this study we have explored the involvement of E-cadherin during receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast differentiation. Osteoclastogenesis involves a period of precursor expansion followed by multiple fusion events to generate a multinuclear osteoclast that is capable of bone resorption. We asked whether E-cadherin participated in early precursor interactions and recognition or was a component of the osteoclast fusion machinery. Here, we show that endogenous E-cadherin expression is the highest during early stages of osteoclast differentiation, with surface expression visible on small precursor cells (fewer than four nuclei per cell) in both RAW 264.7 cells and primary macrophages. Blocking E-cadherin function with neutralizing antibodies prior to the onset of fusion delayed the expression of TRAP, Cathepsin K, DC-STAMP and NFATc1 and significantly diminished multinucleated osteoclast formation. Conversely, E-cadherin-GFP overexpressing macrophages displayed earlier NFATc1 nuclear translocation along with faster formation of multinucleated osteoclasts compared to control macrophages. Through live imaging we identified that disrupting E-cadherin function prolonged the proliferative phase of the precursor population while concomitantly decreasing the proportion of migrating precursors. The lamellipodium and polarized membrane extensions appeared to be the principal sites of fusion, indicating precursor migration was a critical factor contributing to osteoclast fusion. These findings demonstrate that E-cadherin-mediated cell–cell contacts can modulate osteoclast-specific gene expression and prompt differentiating osteoclast precursors toward migratory and fusion activities.     

RANKL is an essential cytokine mediator of polymethylmethacrylate particle-induced osteoclastogenesis.

RANKL is a TNF superfamily member and an essential cytokine mediator of developmental osteoclastogenesis. We examined the role of RANKL in PMMA particle-induced osteoclastogenesis in vitro. In murine whole bone marrow cultures, PMMA particles stimulate a 2.5 fold increase in secreted RANKL, a 5-8 fold increase in osteoclast number and induce the formation of giant multinuclear osteoclasts. RANKL and TNF, potential cytokine mediators of PMMA, had similar osteoclastogenic effects. The RANKL inhibitor OPG was utilized to define the role of RANKL in mediating the PMMA response and was found to inhibit basal and PMMA particle-induced osteoclastogenesis. Additionally, particles stimulate osteoclast formation in RANKL-primed osteoclast precursor cells (devoid of supporting stromal cells) while RANKL untreated osteoclast precursors demonstrate no osteoclastogenic response to particles. Since TNF can potentiate RANKL action and is thought to mediate implant osteolysis we analyzed TNF(-/-) whole bone marrow cultures to elucidate the role of this cytokine. In TNF(-/-) cultures basal osteoclastogenesis remains intact, yet the PMMA effect is blunted. Finally, we show that PMMA, RANKL and TNF all activate the NF-kB and c-jun/AP-1 signaling pathways which are both fundamental to osteoclast formation and are potential sites of signal convergence in RANKL-mediated particle osteoclastogenesis.     

NF-kappaB p50 and p52 expression is not required for RANK-expressing osteoclast progenitor formation but is essential for RANK- and cytokine-mediated osteoclastogenesis.

Expression of RANKL by stromal cells and of RANK and both NF-kappaB p50 and p52 by osteoclast precursors is essential for osteoclast formation. To examine further the role of RANKL, RANK, and NF-KB signaling in this process, we used NF-kappaB p50-/- ;p52-/- double knockout (dKO) and wild-type (WT) mice. Osteoclasts formed in cocultures of WT osteoblasts with splenocytes from WT mice but not from dKO mice, a finding unchanged by addition of RANKL and macrophage colony-stimulating factor (M-CSF). NF-kappaB dKO splenocytes formed more colony-forming unit granulocyte macrophage (CFU-GM) colonies than WT cells, but no osteoclasts were formed from dKO CFU-GM colonies. RANKL increased the number of CFU-GM colonies twofold in WT cultures but not in dKO cultures. Fluorescence-activated cell sorting (FACS) analysis of splenocytes from NF-kappaB dKO mice revealed a two-to threefold increase in the percentage of CD11b (Mac-1) and RANK double-positive cells compared with WT controls. Treatment of NF-kappaB dKO splenocytes with interleukin (IL)-1, TNF-alpha, M-CSF, GM-CSF, and IL-6 plus soluble IL-6 receptor did not rescue the osteoclast defect. No increase in apoptosis was observed in cells of the osteoclast lineage in NF-kappaB dKO or p50-/-;p52+/- (3/4KO) mice. Thus, NF-kappaB p50 and p52 expression is not required for formation of RANK-expressing osteoclast progenitors but is essential for RANK-expressing osteoclast precursors to differentiate into TRAP+ osteoclasts in response to RANKL and other osteoclastogenic cytokines.     

CD40 ligand is essential for generation of specific cytotoxic T cell responses in RNA-pulsed dendritic cell immunotherapy

BACKGROUND: Dendritic cell (DC)-based immunotherapy is a promising form of adjuvant therapy for high-risk tumors. DCs transfected with tumor-associated antigens are capable of stimulating antigen-specific T cells, but cytolytic responses have been disappointing. Activation of DC surface CD40 influences DC cytokine production, particularly that of interleukin (IL)-12, which favors a Th1 (cytotoxic) helper T cell response. This study evaluated the effects of exogenous soluble CD40 ligand (sCD40L) on RNA-transfected DC preparations and their subsequent ability to generate antimelanoma cytolytic T cells. METHODS: Human monocyte-derived DCs were cultured and transfected with mRNA encoding full-length melanoma-associated antigen, Mart-1, and matured with and without sCD40L. DC IL-12 secretion and the ability to stimulate na?ve T cells were assessed by enzyme-linked immunosorbent assay (ELISA), tetramer analysis, Elispot, and (51)Cr release assay. RESULTS: Mature DCs stimulated with sCD40L secreted higher levels of IL-12 compared with immature DCs and DCs matured without sCD40L (P <.001). DCs treated with sCD40L generated a greater number of antigen-specific T cells (P <.05) by tetramer and Elispot analyses, and yielded specific T cells with significant cytotoxicity against HLA-matched melanoma cell lines. CONCLUSIONS: CD40L augments DC IL-12 secretion and is essential to potentiate specific antimelanoma cytolytic responses stimulated by the Mart-1 antigen. sCD40L should be considered a crucial adjuvant in DC preparations for RNA-based DC vaccine therapies.     

Functional expression and microdomain localization of prestin in cultured cells.

INTRODUCTION: Prestin is an essential component of the molecular motor of cochlear outer hair cells that contribute to frequency selectivity and sensitivity of mammalian hearing. A model system to study prestin employs its transfection into cultured HEK 293 cells. Our goal was to characterize prestin's trafficking pathway and localization in the plasma membrane. METHODS: We used immuno-colocalization of prestin with intracellular and plasma membrane markers and sucrose density fractionation to analyze prestin in membrane compartments. Voltage clamping was used to measure nonlinear capacitance (NLC), prestin's electrical signature. RESULTS & DISCUSSION: Prestin targets to the membrane by 24 hours post-transfection when NLC is measurable. Prestin then concentrates into membrane foci that colocalize and fractionate with membrane microdomains. Depleting membrane cholesterol content altered prestin localization and NLC. CONCLUSION: Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.     

CD31 expression on leukocytes is downregulated in vivo during hemodialysis

BACKGROUND/AIM: CD31 on leukocytes is the adhesion molecule involved in the leukocyte extravasation in inflammatory conditions. During hemodialysis with cellulosic membranes, it is considered that activated leukocytes adhere to endothelium, but do not show extravasation. However, it is not elucidated why activated leukocytes do not show endothelial transmigration during hemodialysis with cellulosic membranes. METHODS: In the present study, changes in the expressions of Mac-1 and CD31 on granulocytes and monocytes were analyzed by flow cytometry during hemodialysis in 7 patients treated with regenerated-cellulose (RC) membranes and next with polysulfone (PS) membranes. RESULTS: During dialysis with RC, Mac-1 expressions on granulocytes and monocytes both significantly increased as compared with predialysis values and across the dialyzer. During dialysis with RC, the CD31 expression on granulocytes and monocytes significantly decreased as compared with predialysis values. During dialysis with PS, changes in Mac-1 and CD31 expressions on granulocytes and monocytes were smaller than those during dialysis with RC. CONCLUSIONS: Decreased CD31 expression on leukocytes may affect leukocyte function more in patients chronically hemodialyzed with RC than in those hemodialyzed with PS, since CD31 is important in leukocyte transendothelial migration in inflammatory conditions.     

Colocalization of intracellular osteopontin with CD44 is associated with migration, cell fusion, and resorption in osteoclasts.

Although osteopontin (OPN) is recognized generally as a secreted protein, an intracellular form of osteopontin (iOPN), associated with the CD44 complex, has been identified in migrating fibroblastic cells. Because both OPN and CD44 are expressed at high levels in osteoclasts, we have used double immunofluorescence analysis and confocal microscopy to determine whether colocalization of these proteins has functional significance in the formation and activity of osteoclasts. Analysis of rat bone marrow-derived osteoclasts revealed strong surface staining for CD44 and beta1- and beta3-integrins, whereas little or no staining for OPN or bone sialoprotein (BSP) was observed in nonpermeabilized cells. In permeabilized perfusion osteoclasts and multinucleated osteoclasts, staining for OPN and CD44 was prominent in cell processes, including filopodia and pseudopodia. Confocal microscopy revealed a high degree of colocalization of OPN with CD44 in motile osteoclasts. In cells treated with cycloheximide (CHX), perinuclear staining for OPN and BSP was lost, but iOPN staining was retained within cell processes. In osteoclasts generated from the OPN-null and CD44-null mice, cell spreading and protrusion of pseudopodia were reduced and cell fusion was impaired. Moreover, osteoclast motility and resorptive activity were significantly compromised. Although the area resorbed by OPN-null osteoclasts could be rescued partially by exogenous OPN, the resorption depth was not affected. These studies have identified an intracellular form of OPN, colocalizing with CD44 in cell processes, that appears to function in the formation and activity of osteoclasts.     

Motility-related protein-1/CD9 expression in head and neck squamous cell carcinoma

INTRODUCTION: Motility-related protein (MRP)-1/CD9 is implicated in cell adhesion and motility and was shown to be clearly involved in tumor prognosis and angiogenesis. Elevated MRP-1/CD9 expression on tumor cells has been linked to a favorable prognosis in breast cancer, colon cancer, lung cancer, and HNSCC. Because MRP-1/CD9 is associated with angiogenesis, it might play a role in tumor angiogenesis as well. METHODS: We analyzed MRP-1/CD9 expression in HNSCC specimens and cell lines by real-time RT-PCR and in HNSCC biopsy specimens and stromal vessels by immunohistochemistry. Kruskal Wallis and Chi2 test, univariate and multivariate Cox regression, and Kaplan-Meier methods were used for statistical analysis. RESULTS: Real-time and PCR RT showed elevated expression of MRP-1/CD9 in one (SCC25) of four HNSCC cell lines and two of six HNSCC patients, whereas two cell lines (SCC9 and JPPA) and one HNSCC patient had lower MRP-1/CD9 levels compared with other specimens. Immunohistochemistry demonstrated strong MRP-1/CD9 IR expression on tumor cells in 13 patients (39%), whereas 21 patients (61%) had less to medium MRP-1/CD9 IR expression. Increased MRP-1/CD9 expression on tumor cells was correlated with prolonged patient survival (p =.02) and a longer disease-free interval (p =.004), a diminished recurrence rate (p =.02), and lower stages of neck lymph nodes (p =.04). MRP-1/CD9 IR was also found in a subpopulation of vessels that seem to be less in tumor specimens than in normal mucosa (p <.0001). MRP-1/CD9+ vessels are podoplanin+ and are therefore regarded as lymphatic vessels. CONCLUSIONS: Our results revealed that elevated MRP-1/CD9 expression on HNSCC is linked to a favorable clinical outcome and confirmed reports of MRP-1/CD9 expression in other carcinomas. MRP-1/CD9+ vessels were found to be lymphatic in nature. The number and staining intensity of these vessels is decreased in tumor tissue, which suggests a stabilizing role for this protein in lymphangiogenesis.     

Visceral peritoneum is not essential for solute transport during peritoneal dialysis.

The importance of visceral peritoneum in determining transperitoneal solute exchange was studied by determining the influence of evisceration on diffusive solute transport during peritoneal dialysis. Three series of experiments were performed in anesthetized New Zealand White rabbits. Series 1 studies compared solute transport rates from eviscerated rabbits (N = 5) with those from sham-operated controls (N = 5). Series 2 studies compared solute transport rates from eviscerated (N = 6) and sham-operated rabbits (N = 5) with application of circumferential abdominal compression to control intraperitoneal pressure and presumably maximize dialysate-peritoneum contact. Series 3 studies compared solute transport rates from sham-operated rabbits (N = 4) with and without applied circumferential abdominal compression. Transperitoneal solute exchange of creatinine and FITC-labeled neutral dextran (15 to 40 A) was equally assessed by both the dialysate to plasma concentration ratio at the end of the exchange and the diffusive permeability-area product of the peritoneum. Evisceration reduced creatinine (P less than 0.001) and dextran (15 to 30 A, P less than 0.05) transport to approximately one quarter that of controls in series 1 rabbits. When circumferential abdominal compression was applied in series 2 rabbits, however, evisceration had no effect on peritoneal solute transport rates. Moreover, circumferential abdominal compression per se had no effect on solute exchange in series 3 experiments. These findings demonstrate that the influence of evisceration on peritoneal solute transport depends on the experimental conditions. These observations further demonstrate that visceral peritoneum is not essential for solute transport during peritoneal dialysis.     

Albumin is required for the guinea pig sperm capacitation but is not essential for acrosome reaction and fusion with eggs.

The importance of serum albumin in supporting guinea pig sperm capacitation, acrosome reaction, and fusion with eggs in vitro was studied by incubating the spermatozoa in albumin-free medium containing different synthetic polymers. Serum albumin was found to be an obligatory component in the incubating medium for the capacitation of guinea pig spermatozoa. Albumin in the medium is not essential for the acrosome reaction and fusion with the eggs, but these phenomena take place most efficiently in presence of albumin.     

膀胱移行细胞癌MRP-1/CD9和Ki-67抗原表达及其相关性分析

目的 探讨MRP-1/CD9和Ki-67抗原表达与膀胱移行细胞癌(TCC)生物学行为的关系.方法 膀胱TCC组60例标本经病理复检,UICC临床分期Ta～T1 29例、T2～T4 31例;WHO病理分级G1 19例、G2 14例、G3 27例.其中肿瘤初发41例、复发19例;单发34例、多发26例.11例正常膀胱组织标本作为对照组.应用免疫组化法检测组织MRP-1/CD9和Ki-67抗原表达情况.结果 TCC组MRP-1/CD9阳性表达率48.3%,对照组为90.9%(P=0.023);Ta～T1肿瘤阳性表达率75.9%、T2～T4为22.6%,G1肿瘤阳性表达率73.7%、G2 42.9%、G3 33.3%,随肿瘤分期分级上升,阳性表达率逐渐下降(P=0.000,P=0.024);MRP-1/CD9阳性表达率肿瘤单发者高于多发者(P=0.017),肿瘤初发和复发者之间差异无统计学意义(P=0.511).TCC组Ki-67阳性表达率75.0%,对照组为18.2%(P=0.001);Ta～T1肿瘤阳性表达率62.1%,T2～T4为87.1%;G1肿瘤阳性表达率47.4%、G2 85.7%、G3 88.9%.随着肿瘤分期分级升高,Ki-67阳性表达率逐渐升高(P=0.001,P=0.003).Ki-67阳性表达率肿瘤多发者明显高于单发者(P=0.000),肿瘤初发和复发者之间差异无统计学意义(P=0.083).MRP-1/CD9表达与Ki-67抗原表达呈负相关(r=-2.74,P=0.034).结论 MRP-1/CD9表达与TCC的分期、分级相关,其表达缺失可能是判断该肿瘤预后的一项指标;Ki-67能较准确地评估膀胱TCC的生物学行为,二者可作为膀胱TCC有诊断意义的肿瘤标志物.     

Vascular endothelial growth factor is essential for hyperglycemia-induced structural and functional alterations of the peritoneal membrane.

Long-term peritoneal dialysis is associated with the development of functional and structural alterations of the peritoneal membrane. Long-term exposure to the high glucose concentrations in conventional peritoneal dialysate has been implicated in the pathogenesis of peritoneal hyperpermeability and neoangiogenesis. Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that potently stimulates microvascular permeability and proliferation. High glucose exposure upregulates VEGF expression in various cell types and tissues. This study investigated whether VEGF plays a pathogenetic role in hyperglycemia-induced microvascular dysfunction in the peritoneal membrane. The peritoneal microcirculation of streptozotocin-induced diabetic rats and age-matched controls was studied in vivo with a combination of functional and morphologic techniques. The diabetic microcirculation was characterized by an elevated transport of small solutes, indicating the presence of an increased effective vascular surface area. The leakage of FITC-albumin was more rapid in diabetic vessels, suggesting hyperpermeability for macromolecules. Structurally, an increased vascular density with focal areas of irregular capillary budding was found in the diabetic peritoneum. The hyperglycemia-induced structural and functional microvascular alterations were prevented by long-term treatment with neutralizing anti-VEGF monoclonal antibodies, whereas treatment with isotype-matched control antibodies had no effect. VEGF blockade did not influence microvascular density or macromolecular leakage in control rats, demonstrating specificity for the hyperglycemia-induced alterations. The present results thus support an causative link among high glucose exposure, upregulation of VEGF, and peritoneal microvascular dysfunction.     

Evidence of red blood cell membrane lipid peroxidation in haemodialysis patients

Some metabolic alterations of the pentose-phosphate shunt can increase susceptibility to red blood cell (RBC) lipid peroxidation in uraemic patients on maintenance haemodialysis. We investigated this phenomenon in 19 uraemic patients undergoing chronic haemodialysis by determining RBC malonyldialdehyde (MDA), a secondary product of lipid peroxidation and plasma and RBC tocopherols, which are powerful antioxidants. Evidence of RBC membrane lipid peroxidation was demonstrated by an increase of RBC MDA. RBC tocopherols were significantly decreased because of enhanced antioxidant activity. No significant variations of these parameters were found before and after dialysis.     

Caveolin-1 expression is essential for proper nonshivering thermogenesis in brown adipose tissue

Recently, we have shown that loss of caveolin-1 leads to marked alterations in insulin signaling and lipolysis in white adipose tissue. However, little is known about the role of caveolin-1 in brown adipose tissue (BAT), a tissue responsible for nonshivering thermogenesis. Here, we show that caveolin-1 null mice have a mildly, yet significantly, decreased resting core body temperature. To investigate this in detail, we next subjected the mice to fasting (for 24 h) or cold treatment (4 degrees C for 24 h), individually or in combination. Interestingly, caveolin-1 null mice showed markedly decreased body temperatures in response to fasting or fasting/cold treatment; however, cold treatment alone had no effect. In addition, under these conditions caveolin-1 null mice failed to show the normal increase in serum nonesterified fatty acids induced by fasting or fasting/cold treatment, suggesting that these mice are unable to liberate triglyceride stores for heat production. In accordance with these results, the triglyceride content of BAT was reduced nearly 10-fold in wild-type mice after fasting/cold treatment, but it was reduced only 3-fold in caveolin-1 null mice. Finally, electron microscopy of adipose tissue revealed dramatic perturbations in the mitochondria of caveolin-1 null interscapular brown adipocytes. Taken together, our data provide the first molecular genetic evidence that caveolin-1 plays a critical functional and structural role in the modulation of thermogenesis via an effect on lipid mobilization.     

Red blood cell membrane lipid peroxidation and resistance to erythropoietin therapy in hemodialysis patients.

BACKGROUND: Chronic hemolysis, inadequate production of erythropoietin (EPO) or an impaired response of erythroid stem cells to EPO are the main factors of anemia in end-stage renal disease (ESRD) patients. Oxidative damage of red blood cell (RBC) membrane is a well-established cause of chronic hemolysis in hemodialysis (HD) patients. Administration of high-dose recombinant human EPO (rHuEPO) fails to correct anemia in 5 to 10% HD patients although all established factors of resistance to rHuEPO therapy have been previously ruled out or corrected. PATIENTS AND METHODS: We investigated the degree of RBC membrane oxidative damage in 9 HD patients who failed to respond to maximal rHuEPO administration (more than 200 UI/Kg weekly for 4 months consecutively, group A), compared to 10 patients who showed a good response to standard rHuEPO therapy (group B) and to 10 patients who needed no treatment (group C). RBC malondialdehyde (MDA) was assumed as the index of oxidative stress in erythrocyte membrane. RESULTS: No significant difference in erythrocyte MCV and MCHC, iron status, parathyroid function, aluminum and dialysis-related blood loss was observed between patients of group A, B and C. RBC MDA, reticulocyte count, plasma-free hemoglobin (fhb) and serum lactate dehydrogenase (LDH) were significantly higher while plasma haptoglobin was significantly lower in patients of group A compared to patients of groups B and C. Moreover, a significant inverse relationship was observed between RBC MDA and either plasma hemoglobin, RBC count and hematocrit when all patients were evaluated together. CONCLUSION: In conclusion, increased oxidative damage of RBC membrane is often detectable in HD patients who fail to respond to rHuEPO administration even in the absence of all established factors of resistance to EPO. Peripheral response to rHuEPO may be normal in these patients and persistent anemia may be related to enhanced hemolysis due to oxidative stress. Oxidative damage itself may therefore be considered a factor of resistance to EPO.     

Dissociation of angiogenesis and osteoclastogenesis during endochondral bone formation in neonatal mice.

Invasion of the mineralized matrix by endothelial cells and osteoclasts is a key event in endochondral bone formation. To examine the putative role of osteoclast activity in the angiogenic process, we used two in vivo models of suppressed bone resorption: mice treated with the bisphosphonate clodronate and in osteoclast-deficient, osteopetrotic mice. Angiogenesis was assessed in caudal vertebrae of these neonatal mice. This model enables us to study the interaction between osteoclasts and endothelial cells during endochondral bone formation. In control conditions, sinusoid-like structures were detected in the vicinity of tartrate resistance acid phosphatase positive (TRAcP+) osteoclasts. Treatment with clodronate completely abolished osteoclastic bone resorption, whereas angiogenesis remained unaffected. In line with these observations, in the osteopetrotic mouse mutants c-fos knockout mice and op/op mice, capillaries invaded the calcified cartilage in the absence of osteoclasts. In conclusion, our data strongly suggest that during endochondral bone formation, vascular invasion can occur in the absence of osteo(chondro)clastic resorption. In addition, bisphosphonates show no apparent effect on angiogenesis in this in vivo model. These findings may have important clinical implications in the management of skeletal disorders such as metastatic bone disease, in which both osteoclastic bone resorption and angiogenesis contribute to tumor growth. On the other hand, our results confirm that bisphosphonates can be used safely in the treatment of disorders that affect the growing skeleton, such as in juvenile osteoporosis.